Nitrate levels in water are increasing in the U.S.A, mainly because of runoff from fertilized agricultural land. Hence hazards due to excess nitrate are a consequence of the agricultural industry and are of special interest to E.P.A. At nitrate levels above 50 mg/l, drinking water becomes the main source of exposure to nitrate. Nitrate exposure presents a hazard, because intragastric nitrite can react with amines and amides to form N-nitroso compounds (NNC), which could cause cancer. Ohshima and Bartsch developed a test for measuring in vivo nitrosamine formation, in which proline and nitrate are ingested and N-nitrosoproline (NPRO) is determined in urine. By giving proline without nitrate, this test can be used to measure potential for in vivo nitrosation. This potential depends on intragastric levels of nitrite and nitrosation modifiers, and other factors. The proposed research and future epidemiologic studies will help establish the basis of this test and help answer the questions: Could an elevated exposure to nitrate, especially in drinking water, produce abnormally high levels in vivo of NNC under specified conditions? What nitrate level produces a significant level of NNC? Does this nitrate level vary between countries and with diet? How is NNC formation modified by dietary ascorbate (ASC), vegetables, and macronutrients? Accordingly, we will measure NPRO excretion in the urine of volunteers, after they consume L-proline and varying nitrate levels in drinking water, measure the variability of NPRO excretion, ensure that NPRO is formed endogenously, and determine how nitrate, ASC, vegetables, proteins and other macronutrients influence NPRO formation. These studies (with support from rat experiments) will determine protocols for future epidemiologic studies. We will measure NPRO content of the food, nitrate excretion in urine; nitrate nitrite and thiocyanate levels in saliva; nitrate in drinking water; and nitrate, nitrite and ASC in diet. We will apply these tests to farmers from the Kearney area of Nebraska and to lymphoma patients in remission, who drink high- and low-nitrate water.